2A elements are “self-cleaving” peptides that are derived from viruses 2A elements can be involved in the processing and expression of polyproteins. Without being bound by any particular theory, the presence of the 2A element in the mRNA can cause the translating ribosome to undergo an intra-ribosomal, translational termination-and-restart event during the synthesis of the nascent polypeptide chains. The peptide bond between the first and second polypeptide deriving from the same mRNA is not formed during translation. As a result, when these two polypeptides are liberated from the ribosome, they appear as two separate proteins. Because the apparent effect is as if a single polypeptide had been cleaved by an enzyme post-translationally into two separate polypeptides, for consistency with their historic description, 2A elements may be referred to herein as exemplary “self-cleaving” peptides or as “cleavage sites,” though it is understood that 2A peptides can mediate a ribosomal stop-and-restart event, which may be referred to as a “StopGo” action of the 2A element
Several 2A elements appear to have near 100% cleavage efficiency in their native contexts, but they can be made to cleave at lower efficiencies when they are mutated at particular amino acid residues or introduced into non-native sequences.
Without being bound by any particular theory, surface and secreted soluble proteins and polypeptides of eukaryotic cells can be processed via the secretory pathway. The secretory pathway is described in detail in Alberts et al., Molecular Biology of the Cell, 4th Edition, New York, Garland Science (2002), which is hereby incorporated by reference in its entirety. Typically, in the cytosol of a cell, ribosomes can assemble on polynucleotides that encode polypeptides, for example mRNAs. Ribosomes can mediate the translation of the polynucleotides to produce the encoded polypeptides. The presence of a signal sequence on the polypeptide can mediate the translocation of the polypeptide to the cell's endoplasmic reticulum. The translocation to the endoplasmic reticulum can be co-translational, for example if the signal sequence is located on an N-terminal portion of the polypeptide. Alternatively, the translocation to the endoplasmic reticulum can be post-translational. Thus, while signal sequences are frequently located on N terminal portions of polypeptides, they can also be located internally, or even on a C terminal portion of the polypeptide, and still mediate translocation to the endoplasmic reticulum. Additionally, signal “patches,” can be assembled by particular three-dimensional folding of a polypeptide, and can also mediate translocation to the endoplasmic reticulum. A signal sequence can mediate the polypeptide's entry into the endoplasmic reticulum via one of a plurality of pore in the endoplasmic reticulum membrane. In the absence of an anchor sequence, the entire polypeptide can pass through the pore, and into the lumen of the endoplasmic reticulum. If an anchor sequence is present on the protein or polypeptide, translocation into the endoplasmic reticulum can stop upon the entry of the anchor sequence into the pore, before the entire polypeptide is transported into the endoplasmic reticulum. Accordingly, the protein can remain embedded in the membrane of the endoplasmic reticulum. Luminal and membrane-bound polypeptides in the endoplasmic reticulum can subsequently be transported to the cell's golgi apparatus. Inside the lumen of the endoplasmic reticulum and/or the golgi, the polypeptide, or portions thereof can undergo additional modifications, for example folding, cleavage, and/or glycosylation. From the golgi apparatus, luminal and membrane-bound polypeptides can be transported to the cell membrane, and can be membrane-bound or secreted. Transport between the endoplasmic reticulum, golgi, and cell membrane can be mediated by membrane vesicles. Upon arrival at the cell membrane, membrane vesicles can fuse with the cell membrane. Luminal surfaces of endoplasmic reticulum membrane typically correspond to extracellular surfaces of cell membrane, while cytosolic surfaces of endoplasmic reticulum typically correspond to cytosolic surface of cell membrane. Accordingly, a portion of a transmembrane protein that faces the ER lumen can subsequently face the extracellular environment, and a portion that faces the cytosol can continue to face the cytosol. Cleavage of a luminal portion of a protein or polypeptide from a membrane-bound portion of the polypeptide in the endoplasmic reticulum, golgi, in a membrane vesicle, and/or at the cell surface can allow the cleaved portion to be in the lumen, and/or subsequently secreted from the cell.
B cells are responsible for the production of antibodies in response to foreign antigens. In nature, B cells can produce surface immunoglobulin and secreted antibody from the same immunoglobulin gene via alternative splicing of the pre-messenger RNA.
B cells begin their life in the bone marrow as descendants of the more primitive common hematopoietic stem and progenitor cells. As these cells develop into B cells, they undergo sequential RAG1/2-mediated DNA rearrangement of the heavy and light chain immunoglobulin gene loci in a process called V(D)J rearrangement. Cells that successfully complete this process and assemble a functional B cell receptor (BCR) of the IgM isotype on their surface are able to leave the bone marrow to continue further development in the peripheral lymphoid compartments. The generation of the IgM BCR can be central to B cell development and function, including normal development of B cells, and directing B cell development. In transgenic animals, the provision of a pre-rearranged IgM heavy chain and light chain transgene shuts down the rearrangement of endogenous heavy and light chain genes (allelic exclusion), and guides the ordered development of functional B cells with specificity defined by the transgene.
Mature B cells patrol the body in the general and lymphatic circulations, using their BCRs as antigen sensors. When a cognate antigen engages the BCR, the B cell becomes activated and enters into a germinal center reaction in the lymph node or spleen in a dance of mutual activation with T cells; this process leads to further development into memory B cells or differentiation into antibody-producing plasma cells. The memory B cells will provide a more rapid and higher quality antibody response in the future when the same antigens are encountered again. The plasma cells produce antibodies against the inciting antigens, which leads to their eventual clearance from the body. As B cells differentiate into plasma cells, they switch from producing the membrane-bound IgM BCR to making a soluble, secreted antibody. The genomic machinery for effecting the switch is complex and involves alternative-splicing of the heavy-chain pre-mRNA. The switch replaces the hydrophobic amino acids that form the trans-membrane anchor with a hydrophilic tail that enables the secretion of the BCR as free antibody. The antibody retains the same specificity and isotype as the BCR.